Tear Film Osmometry

ABSTRACT

Osmolarity measurement of a sample fluid, such as tear film, is achieved by depositing an aliquot-sized sample on a sample receiving substrate. The sample fluid is placed on a sample region of the substrate. Energy is imparted to the sample fluid and energy properties of the fluid can be detected to produce a sample fluid reading that indicates osmolarity of the sample fluid. An aliquot-sized volume can comprise, for example, a volume of no more than 20 microliters (μL). The aliquot-sized sample volume can be quickly and easily obtained, even from dry eye sufferers. The imparted energy can comprise electrical, optical or thermal energy. In the case of electrical energy, the energy property of the sample fluid can comprise electrical conductivity. In the case of optical energy, the energy property can comprise fluorescence. In the case of thermal energy, the measured property can be the freezing point of the sample fluid. The substrate can be packaged into a chip, such as by using semiconductor fabrication techniques. An ex vivo osmolarity sensor system that uses the chip can detect energy from the sample region and can provide an accurate osmolarity measurement without user intervention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/358,986 filed Feb. 21, 2006 by Benjamin D. Sullivan, entitled “TearFilm Osmometry”, which is a continuation of co-pending U.S. applicationSer. No. 10/400,617 filed Mar. 25, 2003 by Benjamin D. Sullivan,entitled “Tear Film Osmometry”, now U.S. Pat. No. 7,017,394 issued Mar.28, 2006, which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/401,432 entitled “Volume Independent Tear Film Osmometer”,by Benjamin D. Sullivan, filed Aug. 6, 2002. Priority of the filing dateof these applications is hereby claimed, and the disclosures of theapplications are hereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to measuring the osmoticpressure of fluids and, more particularly, to measuring the osmolarityof tear film.

2. Description of the Related Art

Tears fulfill an essential role in maintaining ocular surface integrity,protecting against microbial challenge, and preserving visual acuity.These functions, in turn, are critically dependent upon the compositionand stability of the tear film structure, which includes an underlyingmucin foundation, a middle aqueous component, and an overlying lipidlayer. Disruption, deficiency, or absence of the tear film can severelyimpact the eye. If unmanaged with artificial tear substitutes or tearfilm conservation therapy, these disorders can lead to intractabledesiccation of the corneal epithelium, ulceration and perforation of thecornea, an increased incidence of infectious disease, and ultimatelypronounced visual impairment and blindness.

Keratoconjunctivitis sicca (KCS), or “dry eye”, is a condition in whichone or more of the tear film structure components listed above ispresent in insufficient volume or is otherwise out of balance with theother components. It is known that the fluid tonicity or osmolarity oftears increases in patients with KCS. KCS is associated with conditionsthat affect the general health of the body, such as Sjogren's syndrome,aging, and androgen deficiency. Therefore, osmolarity of a tear film canbe a sensitive and specific indicator for the diagnosis of KCS and otherconditions.

The osmolarity of a sample fluid (e.g., a tear) can be determined by anex vivo technique called “freezing point depression,” in which solutesor ions in a solvent (i.e. water), cause a lowering of the fluidfreezing point from what it would be without the ions. In the freezingpoint depression analysis, the freezing point of the ionized samplefluid is found by detecting the temperature at which a quantity of thesample (typically on the order of about several milliliters) firstbegins to freeze in a container (e.g., a tube). To measure the freezingpoint, a volume of the sample fluid is collected into a container, suchas a tube. Next, a temperature probe is immersed in the sample fluid,and the container is brought into contact with a freezing bath orPeltier cooling device. The sample is continuously stirred so as toachieve a supercooled liquid state below its freezing point. Uponmechanical induction, the sample solidifies, rising to its freezingpoint due to the thermodynamic heat of fusion. The deviation from thesample freezing point from 0° C. is proportional to the solute level inthe sample fluid. This type of measuring device is sometimes referred toas an osmometer.

Presently, freezing point depression measurements are made ex vivo byremoving tear samples from the eye using a micropipette or capillarytube and measuring the depression of the freezing point that resultsfrom heightened osmolarity. However, these ex vivo measurements areoften plagued by many difficulties. For example, to perform freezingpoint depression analysis of the tear sample, a relatively large volumemust be collected, typically on the order of 20 microliters (μL) of atear film. Because no more than about 10 to 100 nanoliters (nL) of tearsample can be obtained at any one time from a KCS patient, thecollection of sufficient amounts of fluid for conventional ex vivotechniques requires a physician to induce reflex tearing in the patient.Reflex tearing is caused by a sharp or prolonged irritation to theocular surface, akin to when a large piece of dirt becomes lodged inone's eye. Reflex tears are more dilute, i.e. have fewer solute ionsthan the tears that are normally found on the eye. Any dilution of thetear film invalidates the diagnostic ability of an osmolarity test fordry eye, and therefore make currently available ex vivo methodsprohibitive in a clinical setting.

A similar ex vivo technique is vapor pressure osmometry, where a small,circular piece of filter paper is lodged underneath a patient's eyeliduntil sufficient fluid is absorbed. The filter paper disc is placed intoa sealed chamber, whereupon a cooled temperature sensor measures thecondensation of vapor on its surface. Eventually the temperature sensoris raised to the dew point of the sample. The reduction in dew pointproportional to water is then converted into osmolarity. Because of theinduction of reflex tearing and the large volume requirements forexisting vapor pressure osmometers, they are currently impractical fordetermination of dry eye.

The Clifton Nanoliter Osmometer (available from Clifton TechnicalPhysics of Hartford, N.Y., USA) has been used extensively in laboratorysettings to quantify the solute concentrations of KCS patients, but themachine requires a significant amount of training to operate. Itgenerally requires hour-long calibrations and a skilled technician inorder to generate acceptable data. The Clifton Nanoliter Osmometer isalso bulky and relatively expensive. These characteristics seriouslydetract from its use as a clinical osmometer.

In contrast to ex vivo techniques that measure osmolarity of tearsamples removed from the ocular surface, an in vivo technique thatattempted to measure osmolarity directly on the ocular surface used apair flexible pair of electrodes that were placed directly underneaththe eyelid of the patient. The electrodes were then plugged into an LCRmeter to determine the conductivity of the fluid surrounding them. Whileit has long been known that conductivity is directly related to theionic concentration, and hence osmolarity of solutions, placing thesensor under the eyelid for half a minute likely induced reflex tearing.Furthermore, these electrodes were difficult to manufacture and posedincreased health risks to the patient as compared to simply collectingtears with a capillary.

It should be apparent from the discussion above that current osmolaritymeasurement techniques are unavailable in a clinical setting and can'tattain the volumes necessary for dry eye patients. Thus, there is a needfor an improved, clinically feasible, nanoliter-scale osmolaritymeasurement. The present invention satisfies this need.

SUMMARY

Osmolarity measurement of a sample fluid, such as a tear film, isachieved by depositing an aliquot volume of the sample fluid on amicrochip having a substrate and a sample region of the substrate,wherein the volume of the sample fluid operatively covers a sufficientportion of the sample region such that energy imparted to the samplefluid is detected from the sample region to produce an output signalthat indicates osmolarity of the sample fluid. Thus, an osmolaritymeasurement of the sample fluid can be obtained from the detected energyof the sample volume. The aliquot-sized sample volume can be quickly andeasily obtained, even from dry eye patients. An aliquot volume cancomprise, for example, a volume of no more than 20 microliters (μL), butcan be as little as 1 nL. An osmolarity sensor system can receive themicrochip and sample volume, and can detect energy from the samplevolume to display an accurate osmolarity measurement. In this way, areliable osmolarity measurement can be obtained with minimuminconvenience and discomfort to a patient, without requiring a greatdeal of skill to obtain the measurement, and with a high degree ofrepeatability and accuracy.

The sample fluid volume can be easily deposited on the substrate sampleregion. Energy is transferred to the sample fluid such that energyproperties of the sample fluid can be detected to provide an accuratemeasurement of sample osmolarity. The energy transferred can compriseelectrical energy. For example, electrodes of the substrate can bespaced such that an aliquot-sized sample volume can bridge at least twoof the electrodes. Electrical energy passing through the electrodes canbe used to measure conductivity and thereby provide an osmolaritymeasure. The energy transferred can comprise optical energy. Forexample, nanometer-sized spheres can be coated with luminescention-sensitive chemicals. When the spheres are exposed to a tear filmsample and are excited with light energy such as laser light, thespheres will luminesce such that the emitted light can be correlated toosmolarity of the sample. The energy transferred can comprise thermalenergy. Continuous cooling of the sample results in a reducedconductivity of the sample upon freezing, which allows correlation ofthe determined freezing point with the osmolarity of the sample.

An osmolarity sensor system for measuring osmolarity of a sample fluidincludes a sample fluid reception device and a platform for datacommunication. The sample fluid reception device can be produced, forexample, using semiconductor fabrication techniques. Microprocessorfabrication techniques allow the reception device to be as simple as aset of electrodes printed on a microchip, or as complicated as alogic-enabled microprocessor capable of enacting measurement dynamics onthe sample fluid reception element. Microfabrication also enablestemperature sensing and temperature control directly on the sample fluidreception device. The platform for data communication receives outputfrom the sample fluid reception device, and interprets and displays thisinformation as an osmolarity of the sample fluid to the user via LCD orequivalent display mechanism.

Other features and advantages of the present invention should beapparent from the following description of the preferred embodiment,which illustrates, by way of example, the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an aliquot-sized sample receiving chip for measuringthe osmolarity of a sample fluid.

FIG. 2 illustrates an alternative embodiment of a sample receiving chipthat includes a circuit region with an array of electrodes imprintedwith photolithography techniques.

FIG. 3 illustrates another alternative embodiment of the FIG. 1 chip,wherein a circuit region includes printed electrodes arranged in aplurality of concentric circles.

FIG. 4 is a top view of the chip shown in FIG. 2.

FIG. 5 is a top view of the chip shown in FIG. 3.

FIG. 6 is a block diagram of an osmolarity measurement system configuredin accordance with the present invention.

FIG. 7 is a perspective view of a tear film osmolarity measurementsystem constructed in accordance with the present invention.

FIG. 8 is a side section of the sample receiving chip showing theopening in the exterior packaging.

FIG. 9 is a calibration curve relating the sodium content of the samplefluid with electrical conductivity.

FIG. 10 illustrates a hinged base unit of the osmometer that utilizesthe sample receiving chips described in FIGS. 1-5.

FIG. 11 illustrates a probe card configuration for the sample receivingchip and processing unit.

FIG. 12 illustrates an optical osmolarity measurement system constructedin accordance with the present invention.

FIG. 13 is a flowchart describing an exemplary osmolarity measurementtechnique in accordance with the invention.

DETAILED DESCRIPTION

Exemplary embodiments are described for measuring the osmolarity of analiquot volume of a sample fluid (e.g., tear film, sweat, blood, orother fluids). The exemplary embodiments are configured to be relativelyfast, non-invasive, inexpensive, and easy to use, with minimal injury ofrisk to the patient. Accurate measurements can be provided with aslittle as nanoliter volumes of a sample fluid. For example, a measuringdevice configured in accordance with the invention enables osmolaritymeasurement with no more than 20 μL of sample fluid, and typically muchsmaller volumes can be successfully measured. In one embodimentdescribed further below, osmolarity measurement accuracy is notcompromised by variations in the volume of sample fluid collected, sothat osmolarity measurement is substantially independent of collectedvolume. The sample fluid can include tear film, sweat, blood, or otherbodily fluids. It should be noted, however, that sample fluid cancomprise other fluids, such as milk or other beverages.

FIG. 1 illustrates an exemplary embodiment of an osmolarity chip 100that can be used to measure the osmolarity of a sample fluid 102, suchas a tear film sample. In the FIG. 1 embodiment, the chip 100 includes asubstrate 104 with a sample region having sensor electrodes 108, 109 andcircuit connections 110 imprinted on the substrate. The electrodes andcircuit connections are preferably printed using well-knownphotolithographic techniques. For example, current techniques enable theelectrodes 108, 109 to have a diameter in the range of approximately one(1) to eighty (80) microns, and spaced apart sufficiently so that noconductive path exists in the absence of sample fluid. Currentlyavailable techniques, however, can provide electrodes of less than onemicron in diameter, and these are sufficient for a chip constructed inaccordance with the invention. The amount of sample fluid needed formeasurement is no more than is necessary to extend from one electrode tothe other, thereby providing an operative conductive path. Thephotolithographic scale of the chip 100 permits the measurement to bemade for aliquot-sized samples in a micro- or nano-scale level. Forexample, reliable osmolarity measurement can be obtained with a samplevolume of less than 20 μL of tear film. A typical sample volume is lessthan one hundred nanoliters (100 nL). It is expected that it will berelatively easy to collect 10 nL of a tear film sample even frompatients suffering from dry eye.

The chip 100 is configured to transfer energy to the sample fluid 102and enable detection of the sample fluid energy properties. In thisregard, a current source is applied across the electrodes 108, 109through the connections 110. The osmolarity of the sample fluid can bemeasured by sensing the energy transfer properties of the sample fluid102. The energy transfer properties can include, for example, electricalconductivity, such that the impedance of the sample fluid is measured,given a particular amount of electrical power (e.g., current) that istransferred into the sample through the connections 110 and theelectrodes 108, 109.

If conductivity of the sample fluid is to be measured, then preferably asinusoidal signal on the order of ten volts at approximately 10 kHz isapplied. The real and imaginary parts of the complex impedance of thecircuit path from one electrode 108 through the sample fluid 102 to theother electrode 109 are measured. At the frequencies of interest, it islikely that the majority of the electrical signal will be in the realhalf of the complex plane, which reduces to the conductivity of thesample fluid. This electrical signal (hereafter referred to asconductivity) can be directly related to the ion concentration of thesample fluid 102, and the osmolarity can be determined. Moreover, if theion concentration of the sample fluid 102 changes, the electricalconductivity and the osmolarity of the fluid will change in acorresponding manner. Therefore, the osmolarity is reliably obtained. Inaddition, because the impedance value does not depend on the volume ofthe sample fluid 102, the osmolarity measurement can be madesubstantially independent of the sample volume.

As an alternative to the input signal described above, more complexsignals can be applied to the sample fluid whose response willcontribute to a more thorough estimate of osmolarity. For example,calibration can be achieved by measuring impedances over a range offrequencies. These impedances can be either simultaneously (via combinedwaveform input and Fourier decomposition) or sequentially measured. Thefrequency versus impedance data will provide information about thesample and the relative performance of the sample fluid measurementcircuit.

FIG. 2 illustrates an alternative embodiment of a sample receiving chip200 that measures osmolarity of a sample fluid 202, wherein the chipcomprises a substrate layer 204 with a sample region 206 comprising animprinted circuit that includes an array of electrodes 208. In theillustrated embodiment of FIG. 2, the sample region 206 has a 5-by-5array of electrodes that are imprinted with photolithographictechniques, with each electrode 208 having a connection 210 to one sideof the substrate 204. Not all of the electrodes 208 in FIG. 2 are shownwith a connection, for simplicity of illustration. The electrodesprovide measurements to a separate processing unit, described furtherbelow.

The electrode array of FIG. 2 provides a means to measure the size ofthe tear droplet 202 by detecting the extent of conducting electrodes208 to thereby determine the extent of the droplet. In particular,processing circuitry can determine the number of electrodes that areconducting, and therefore the number of adjacent electrodes that arecovered by the droplet 202 will be determined. The planar area of thesubstrate that is covered by the sample fluid is thereby determined.With a known nominal surface tension of the sample fluid, the height ofthe sample fluid volume over the planar area can be reliably estimated,and therefore the volume of the droplet 202 can be determined.

FIG. 3 illustrates another alternative embodiment of a sample receivingchip 300 on which a sample fluid 302 is deposited. The chip comprises asubstrate layer 304, wherein a sample region 306 is provided withelectrodes 308 that are configured in a plurality of concentric circles.In a manner similar to the square array of FIG. 2, the circulararrangement of the FIG. 3 electrodes 308 also provides an estimate ofthe size of the sample fluid volume 302 because the droplet typicallycovers a circular or oval area of the sample region 302. Processingcircuitry can detect the largest (outermost) circle of electrodes thatare conducting, and thereby determine a planar area of coverage by thefluid sample. As before, the determined planar area provides a volumeestimate, in conjunction with a known surface tension and correspondingvolume height of the sample fluid 302. In the FIG. 3 illustratedembodiment, the electrodes 308 can be printed using well-knownphotolithography techniques that currently permit electrodes to have adiameter in the range of one (1) to eighty (80) microns. This allows thesub-microliter droplet to substantially cover the electrodes. Theelectrodes can be printed over an area sized to receive the samplefluid, generally covering 1 mm2 to 1 cm2.

The electrodes and connections shown in FIG. 1, FIG. 2, and FIG. 3 canbe imprinted on the respective substrate layers as electrodes withcontact pads, using photolithographic techniques. For example, theelectrodes can be formed with different conductive metalization such asaluminum, platinum, titanium, titanium-tungsten, and other similarmaterial. In one embodiment, the electrodes can be formed with adielectric rim to protect field densities at the edges of theelectrodes. This can reduce an otherwise unstable electric field at therim of the electrode.

Top views of the exemplary embodiments of the chips 200 and 300 areillustrated in FIG. 4 and FIG. 5, respectively. The embodiments show thedetailed layout of the electrodes and the connections, and illustratehow each electrode can be electrically connected for measuring theelectrical properties of a sample droplet. As mentioned above, thelayout of the electrodes and the connections can be imprinted on thesubstrate 100, 200, 300 using well-known photolithographic techniques.

FIG. 6 is a block diagram of an osmometry system 600 configured inaccordance with an embodiment of the present invention, showing howinformation is determined and used in a process that determinesosmolarity of a sample fluid. The osmometry system 600 includes ameasurement device 604 and a processing device 606. The measurementdevice receives a volume of sample fluid from a collection device 608.The collection device can comprise, for example, a micropipette orcapillary tube. The collection device 608 collects a sample tear film ofa patient, such as by using negative pressure from a fixed-volumemicropipette or charge attraction from a capillary tube to draw a smalltear volume from the vicinity of the ocular surface of a patient.

The measurement device 604 can comprise a system that transfers energyto the fluid in the sample region and detects the imparted energy. Forexample, the measurement device 604 can comprise circuitry that provideselectrical energy in a specified waveform (such as from a functiongenerator) to the electrical path comprising two electrodes bridged bythe sample fluid. The processing device 606 detects the energy impartedto the sample fluid and determines osmolarity. The processing device cancomprise, for example, a system including an RLC multimeter thatproduces data relating to the reactance of the fluid that forms theconductive path between two electrodes, and including a processor thatdetermines osmolarity through a table look-up scheme. If desired, theprocessing device can be housed in a base unit that receives one of thechips described above.

As mentioned above, a sample sufficient to provide an osmolaritymeasurement can contain less than 20 microliters (μL) of fluid. Atypical sample of tear film in accordance with the invention iscollected by a fluid collector such as a capillary tube, which oftencontains less than one microliter of tear film. Medical professionalswill be familiar with the use of micropipettes and capillary tubes, andwill be able to easily collect the small sample volumes describedherein, even in the case of dry eye sufferers.

The collected sample fluid is expelled from the collection device 608 tothe measurement device 604. The collection device can be positionedabove the sample region of the chip substrate either manually by amedical professional or by being mechanically guided over the sampleregion. In one embodiment, for example, the collection device (e.g., acapillary tube) is mechanically guided into position with aninjection-molded plastic hole in a base unit, or is fitted to a set ofclamps with precision screws (e.g., a micromanipulator with needles formicrochip interfaces). In another embodiment, the guide is acomputer-guided feedback control circuitry that holds the capillary tubeand automatically lowers it into the proper position. The electrodes andconnections of the chips measure energy properties of the sample fluid,such as conductivity, and enable the measured properties to be receivedby the processing device 606. The measured energy properties of thesample fluid include electrical conductivity and can also include otherparameters, such as both parts of the complex impedance of the sample,the variance of the noise in the output signal, and the measurementdrift due to resistive heating of the sample fluid. The measured energyproperties are processed in the processing device 606 to provide theosmolarity of the sample. In one embodiment, the processing device 606comprises a base unit that can accept a chip and can provide electricalconnection between the chip and the processing device 606. In anotherembodiment, the base unit can include a display unit for displayingosmolarity values. It should be noted that the processing device 606and, in particular, the base unit can be a hand-held unit.

FIG. 7 is a perspective view of a tear film osmolarity measuring system700 constructed in accordance with the present invention. In theillustrated embodiment of FIG. 7, the exemplary system 700 includes ameasuring unit 701 that comprises a chip, such as one of the chipsdescribed above, and a connector or socket base 710, which provides theappropriate measurement output. The system 700 determines osmolarity bymeasuring electrical conductivity of the sample fluid. Therefore, themeasurement chip 701 comprises a semiconductor integrated circuit (IC)chip with a substrate having a construction similar to that of the chipsdescribed above in connection with FIG. 1 through FIG. 5. Thus, the chip701 includes a substrate layer with a sample region that is defined byat least two electrodes printed onto the substrate layer (such detailsare of a scale too small to be visible in FIG. 7; see FIG. 1 throughFIG. 5). The substrate and sample region are encased within an inertpackage, in a manner that will be known to those skilled in the art. Inparticular, the chip 701 is fabricated using conventional semiconductorfabrication techniques into an IC package 707 that includes electricalconnection legs 708 that permit electrical signals to be received by thechip 701 and output to be communicated outside of the chip. Thepackaging 707 provides a casing that makes handling of the chip moreconvenient and helps reduce evaporation of the sample fluid.

FIG. 8 shows that the measurement chip 701 is fabricated with anexterior opening hole 720 into which the sample fluid 702 is inserted.Thus, the hole 720 can be formed in the semiconductor packaging 707 toprovide a path through the chip exterior to the substrate 804 and thesample region 806. The collection device (such as a micropipette orcapillary tube) 808 is positioned into the hole 720 such that the samplefluid 702 is expelled from the collection device directly onto thesample region 806 of the substrate 804. The hole 720 is sized to receivethe tip of the collection device. The hole 720 forms an opening orfunnel that leads from the exterior of the chip onto the sample region806 of the substrate 804. In this way, the sample fluid 702 is expelledfrom the collection device 808 and is deposited directly on the sampleregion 806 of the substrate 804. The sample region is sized to receivethe volume of sample fluid from the collection device. In FIG. 8, forexample, the electrodes form a sample region 806 that is generally in arange of approximately 1 mm2 to 1 cm2 in area.

Returning to FIG. 7, the chip 701 can include processing circuitry 704that comprises, for example, a function generator that generates asignal of a desired waveform, which is applied to the sample regionelectrodes of the chip, and a voltage measuring device to measure theroot-mean-square (RMS) voltage value that is read from the chipelectrodes. The function generator can produce high frequencyalternating current (AC) to avoid undesirable direct current (DC)effects for the measurement process. The voltage measuring device canincorporate the functionality of an RLC measuring device. Thus, the chip701 can incorporate the measurement circuitry as well as the sampleregion electrodes. The processing circuitry can include a centralprocessing unit (CPU) and associated memory that can store programminginstructions (such as firmware) and also can store data. In this way, asingle chip can include the electrodes and associated connections forthe sample region, and on a separate region of the chip, can alsoinclude the measurement circuitry. This configuration will minimize theassociated stray resistances of the circuit structures.

As noted above, the processing circuitry 704 applies a signal waveformto the sample region electrodes. The processing circuitry also receivesthe energy property signals from the electrodes and determines theosmolarity value of the sample fluid. For example, the processing unitreceives electrical conductivity values from a set of electrode pairs.Those skilled in the art will be familiar with techniques and circuitryfor determining the conductivity of a sample fluid that forms aconducting path between two or more electrodes.

In the FIG. 7 embodiment, the processing unit 704 produces signalwaveforms at a single frequency, such as 100 kHz and 10 Voltspeak-to-peak. The processing circuitry 704 then determines theosmolarity value from the sodium content correlated to the electricalconductivity using a calibration curve, such as the curve shown in FIG.9. In this case, the calibration curve is constructed as a transferfunction between the electrical conductivity (voltage) and theosmolarity value (i.e., the sodium content). It should be noted,however, that other calibration curves can also be constructed toprovide transfer functions between other energy properties and theosmolarity value. For example, the variance, autocorrelation and driftof the signal can be included in an osmolarity calculation. If desired,the osmolarity value can also be built upon multi-variable correlationcoefficient charts or neural network interpretation so that theosmolarity value can be optimized with an arbitrarily large set ofmeasured variables.

In an alternate form of the FIG. 7 embodiment, the processing unit 704produces signal waveforms of a predetermined frequency sweep, such as 1kHz to 100 kHz in 1 kHz increments, and stores the conductivity andvariance values received from the set of electrode pairs at eachfrequency. The output signal versus frequency curve can then be used toprovide higher order information about the sample which can be used withthe aforementioned transfer functions to produce an ideal osmolarityreading.

As shown in FIG. 7, the base socket connector 710 receives the pins 708of the chip 701 into corresponding sockets 711. The connector 710, forexample, can supply the requisite electrical power to the processingcircuitry 704 and electrodes of the chip. Thus, the chip 701 can includethe sample region electrodes and the signal generator and processingcircuitry necessary for determining osmolarity, and the outputcomprising the osmolarity value can be communicated off the chip via thepins 708 through the connector 710 and to a display readout.

If desired, the base connector socket 710 can include a Peltier layer712 located beneath the sockets that receive the pins 708 of the chip701. Those skilled in the art will understand that a Peltier layercomprises an electrical/ceramic junction such that properly appliedcurrent can cool or heat the Peltier layer. In this way, the sample chip701 can be heated or cooled, thereby further controlling evaporation ofthe sample fluid. It should be apparent that evaporation of the samplefluid should be carefully controlled, to ensure accurate osmolarityvalues obtained from the sample fluid.

FIG. 10 shows an alternative embodiment of an osmometer in which thechip does not include an on-chip processing unit such as describedabove, but rather includes limited circuitry comprising primarily thesample region electrodes and interconnections. That is, the processingunit is separately located from the chip and can be provided in the baseunit.

FIG. 10 shows in detail an osmometer 1000 that includes a base unit1004, which houses the base connector 710, and a hinged cover 1006 thatcloses over the base connector 710 and a received measurement chip 701.Thus, after the sample fluid has been dispensed on the chip, the chip isinserted into the socket connector 710 of the base unit 1004 and thehinged cover 1006 is closed over the chip to reduce the rate ofevaporation of the sample fluid.

It should be noted that the problem with relatively fast evaporation ofthe sample fluid can generally be handled in one of two ways. One way isto measure the sample fluid voltage quickly as soon possible after thedroplet is placed on the sample region of the chip. Another way is toenable the measuring unit to measure the rate of evaporation along withthe corresponding changes in conductivity values. The processing unitcan then post-process the output to estimate the osmolarity value. Theprocessing can be performed in the hardware or in software stored in thehardware. Thus, the processing unit can incorporate different processingtechniques such as using neural networks to collect and learn aboutcharacteristics of the fluid samples being measured for osmolarity, aswell as temperature variations, volume changes, and other relatedparameters so that the system can be trained in accordance with neuralnetwork techniques to make faster and more accurate osmolaritymeasurements.

FIG. 11 shows another alternative construction, in which the osmolaritysystem utilizes a sample receiving chip 1102 that does not include ICpackaging such as shown in FIG. 7. Rather, the FIG. 11 measurement chip1102 is configured as a chip with an exposed sample region comprisingthe electrodes and associated connections, but the processing circuitryis located in the base unit for measuring the energy properties of thesample fluid. In this alternative construction, a connector similar tothe connector socket 710 allows transmission of measured energyproperties to the processing unit in the base unit. Those skilled in theart will understand that such a configuration is commonly referred to aprobe card structure.

FIG. 11 shows a probe card base unit 1100 that receives a sample chipprobe card 1102 that comprises a substrate 1104 with a sample region1106 on which are formed electrodes 1108 that are wire bonded to edgeconnectors 1110 of the probe card. When the hinged lid 1112 of the baseunit is closed down over the probe card, connecting tines 1114 on theunderside of the lid come into mating contact with the edge connectors1110. In this way, the electrodes of the sample region 1106 are coupledto the processing circuitry and measurement can take place. Theprocessing circuitry of the probe card embodiment of FIG. 11 can beconfigured in either of the configurations described above. That is, theprocessing to apply current to the electrodes and to detect energyproperties of the sample fluid and determine osmolarity can be locatedon-chip, on the substrate of the probe card 1102, or the processingcircuitry can be located off-chip, in the base unit 1100.

In all the alternative embodiments described above, the osmometer isused by placing a new measurement chip into the base unit while thehinged top is open. Upon placement into the base unit, the chip ispowered up and begins monitoring its environment. Recording outputsignals from the chip at a rate of, for example, 1 kHz, will fullycapture the behavior of the system. Placing a sample onto any portion ofthe electrode array generates high signal-to-noise increase inconductivity between any pair of electrodes covered by the sample fluid.The processing unit will recognize the change in conductivity as beingdirectly related to the addition of sample fluid, and will beginconversion of electronic signals into osmolarity data once this type ofchange is identified. This strategy occurs without intervention bymedical professionals. That is, the chip processing is initiated uponcoupling to the base unit and is not dependent on operating the lid ofthe base unit or any other user intervention.

In any of the configurations described above, either the “smart chip”with processing circuitry on-chip (FIG. 7), or the electrode-onlyconfiguration with processing circuitry off-chip (FIG. 10), in apackaged chip (FIG. 7 and FIG. 10) or in a probe card (FIG. 11), thesample receiving chip can be disposed of after each use, so that thebase unit serves as a platform for interfacing with the disposablemeasurement chip. As noted, the base unit can also include relevantcontrol, communication, and display circuits (not shown), as well assoftware, or such features can be provided off-chip in the base unit. Inthis regard, the processing circuitry can be configured to automaticallyprovide sufficient power to the sample region electrodes to irreversiblyoxidize them after a measurement cycle, such that the electrodes arerendered inoperable for any subsequent measurement cycle. Upon inserteda used chip into the base unit, the user will be given an indicationthat the electrodes are inoperable. This helps prevent inadvertentmultiple use of a sample chip, which can lead to inaccurate osmolarityreadings and potentially unsanitary conditions.

A secondary approach to ensure that a previously used chip is not placedback into the machine includes encoding serial numbers, or codesdirectly onto the chip. The base unit will store the used chip numbersin memory and cross-reference them against new chips placed in the baseconnector. If the base unit finds that the serial number of the usedchip is the same as an old chip, then the system will refuse to measureosmolarity until a new chip is inserted. It is important to ensure useof a new chip for each test because proteins adsorb and salt crystalsform on the electrodes after evaporation has run its course, whichcorrupt the integrity of the measuring electrodes.

In a further embodiment shown in FIG. 12, the osmolarity of a samplefluid can be measured optically in an optical measurement system 1200 byusing optical indicators 1202 disposed on a measuring region 1212 of thechip substrate 1204. The optical indicators 1202 can comprise, forexample, nano-scale spheres, also called nanobeads, that are coated withchemicals whose fluorescence varies with exposure to sample fluid ofvarying osmolarity, i.e. ionophores. The nanobeads 1202 can be depositedon the chip substrate 1204 on top of the electrodes described above forthe conductivity-measuring chips. The electrodes are useful fordetermining the volume of the sample fluid, as described above. However,other volume-measuring elements may be used to determine the volume ofthe sample fluid. Preferably, the optical chip is produced with inertpackaging such as described above in connection with FIG. 7, including achip opening hole through which the collection device tip can beinserted. The sample fluid is then expelled from the collection deviceand the sample fluid comes into contact with a predetermined, fixednumber of the nanobeads per electrode site, which become immersed in thesample fluid.

When the nanobeads 1202 are illuminated with an optical energy source1210, such as a laser, the beads 1202 will fluoresce in accordance withthe osmolarity of the sample fluid 1206. The fluorescence can bedetected using a suitable optical detector light receiving device 1208,such as a conventional charge-coupled device (CCD) array, photodiode, orthe like. The resulting output signal of the light receiving array canindicate the osmolarity value of the sample fluid. It should be notedthat the nano-scale beads are sized such that an aliquot-sized fluidsample 1206 (i.e., no more than 20 microliters of the fluid) willordinarily produce sufficient fluorescence to provide an output signalthat can be detected by the light receiving device 1208 and that canindicate osmolarity of the sample fluid. The amount of fluorescence canbe normalized by calculating how many nanobeads were activated by fluid,by measuring which electrode pairs were activated by the sample fluid.This normalization accounts for the sample volume and allows the volumeindependence feature of the prior embodiment to be retained.

FIG. 13 is a flowchart describing an exemplary osmolarity measurementtechnique in accordance with the invention. A body fluid sample, such asa tear film, is collected at box 1300. The sample typically containsless than one microliter. At box 1302, the collected sample is depositedon a sample region of the chip substrate. The energy properties of thesample are then measured at box 1304. The measured energy properties arethen processed, at box 1306, to determine the osmolarity of the sample.If the chip operates in accordance with electrical conductivitymeasurement, then the measurement processing at box 1306 can include the“electrode oxidation” operation described above that renders the chipelectrodes inoperable for any subsequent measuring cycles.

In the measurement process for a conductivity measuring system, asubstantially instantaneous shift is observed from the open circuitvoltage to a value that closely represents the state of the sample atthe time of collection, upon placement of a sample tear film on anelectrode array of the substrate. Subsequently, a drift in theconductivity of the sample will be reflected as a continual change inthe output.

The output of the measurement chip can be a time-varying voltage that istranslated into an osmolarity value. Thus, in a conductivity-basedsystem, more information than just the “electrical conductivity” of thesample can be obtained by measuring the frequency response over a widerange of input signals, which improves the end stage processing. Forexample, the calibration can be made over a multiple frequencies (e.g.,measure ratio of signals at 10, 20, 30, 40, 50, 100 Hz) to make themeasurement process a relative calculation. This makes the chip-to-chipvoltage drift small. The standard method for macroscale electrode basedmeasurements (i.e. in a pH meter, or microcapillary technique) is torely upon known buffers to set up a linear calibration curve. Becausephotolithography is an extremely reproducible manufacturing technique,when coupled to a frequency sweep, calibration can be performed withoutoperator intervention.

As mentioned above, the processing of the energy properties can beperformed in a neural network configuration, where the seeminglydisparate measured data points obtained from the energy properties canbe used to provide more accurate osmolarity reading than from a singleenergy property measurement. For example, if only the electricalconductivity of the sample is measured, then the calibration curve canbe used to simply obtain the osmolarity value corresponding to theconductivity. This osmolarity value, however, generally will not be asaccurate as the output of the neural network.

The neural network can be designed to operate on a collection ofcalibration curves that reflects a substantially optimized transferfunction between the energy properties of the sample fluid and theosmolarity. Thus, in one embodiment, the neural network constructs acollection of calibration curves for all variables of interest, such asvoltage, evaporation rate and volume change. The neural network can alsoconstruct or receive as an input a priority list that assigns animportance factor to each variable to indicate the importance of thevariable to the final outcome, or the osmolarity value. The neuralnetwork constructs the calibration curves by training on examples ofreal data where the final outcome is known a priori. Accordingly, theneural network will be trained to predict the final outcome from thebest possible combination of variables. This neural networkconfiguration that processes the variables in an efficient combinationis then loaded into the processing unit residing in the measurement chip701 or the base unit. Once trained, the neural network can be configuredin software or hardware.

Although the embodiments described above for measuring osmolarityprovides substantial advantage over the conventional osmolaritymeasuring techniques such as a freezing point depression technique, theteachings of the present invention can be used to determine osmolarityof a sample in accordance with the freezing point depression technique.Accordingly, the exemplary osmometry system 600 of FIG. 6 can be used toprovide an osmolarity value based on the freezing point depressiontechnique.

The freezing point depression system involves collecting and depositingthe sample fluid in a similar manner as in the boxes 1300 and 1302 ofthe flowchart in FIG. 13. As noted above, however, the osmometer of theosmometer system can include a cooling device, such as a Peltier coolingdevice. In the FIG. 7 embodiment described above, the Peltier device isdisposed on the socket 710 or the chip 701 (see FIG. 7) to cool thesample. If desired, the Peltier cooling device can be used to cool thesample fluid to the freezing point of the sample fluid. Aphoto-lithographed metal junction, or p-n junction, known as athermocouple, can be used to monitor the temperature of aliquot-sizedsamples. The thermocouple would operate in parallel to the electrodearray and Peltier cooling device, where the chip would be cooled belowfreezing so that the sample becomes a solid. Upon solidification, theelectrical conductivity of the sample will drastically change. Becausethe thermocouple is continually measuring the temperature, the point atwhich the conductivity spikes can be correlated to the depressedfreezing point. Alternatively, the chip could be supercooled immediatelyprior to sample introduction by the Peltier unit, and then by using theresistive heating inherent to the electrodes, a current can be passedalong the solid phase material. Upon melting, the conductivity willagain drastically change. In the second measurement technique, it islikely that evaporation will be less of a factor. Thus, the presentinvention permits freezing point depression to be performed atsignificantly smaller volumes of sample fluid than previously possible.

The present invention has been described above in terms of exemplaryembodiments so that an understanding of the present invention can beconveyed. Any embodiment described herein as “exemplary” is notnecessarily to be construed as preferred or advantageous over otherembodiments. Moreover, there are many configurations for the osmometerand associated components not specifically described herein but withwhich the present invention is applicable. The present invention shouldtherefore not be seen as limited to the particular embodiments describedherein, but rather, it should be understood that the present inventionhas wide applicability with respect to tear film osmometry generally.All modifications, variations, or equivalent arrangements andimplementations that are within the scope of the attached claims shouldtherefore be considered within the scope of the invention.

1. A sample receiving chip comprising: a substrate that receives analiquot volume of a sample fluid; a sample region of the substrate,sized such that the volume of the sample fluid is sufficient tooperatively cover a portion of the sample region, whereupon energytransfer properties of the sample fluid can be detected from the sampleregion in response to imparting energy into the sample fluid to producean electrical signal comprising a sample fluid reading, wherein thesample fluid reading is directly related to the sample fluid energytransfer properties and the sample fluid reading directly determinesosmolarity of the sample fluid.
 2. A chip as defined in claim 1, whereinthe sample region includes a plurality of electrodes disposed to contactthe sample.
 3. A chip as defined in claim 2, wherein the plurality ofelectrodes is arranged in a row and column array.
 4. A chip as definedin claim 2, further comprising a plurality of conductive connectionlines coupled to the plurality of electrodes, wherein the conductiveconnection lines provide means for transferring energy to and from thesample fluid.
 5. A chip as defined in claim 4, further comprising: aprocessing unit configured to receive energy properties of the samplefluid from the plurality of conductive connection lines, wherein theprocessing unit processes the received energy properties and outputs theosmolarity of the sample fluid.
 6. A chip as defined in claim 1, whereinarea of the sample region on the substrate is less than about onecentimeter square.
 7. A chip as defined in claim 1, further comprising:a temperature control element in communication with the substrate.
 8. Achip as defined in claim 7, wherein the temperature control elementincludes a Peltier cooling device.
 9. An osmolarity measuring system formeasuring osmolarity of a sample fluid, the system comprising: ameasurement device comprising a sample receiving chip that includes asubstrate having a sample region configured to contact the sample fluidto produce an electrical signal in response to imparting energy into thesample fluid such that the electrical signal comprises a sample fluidreading that is directly related to energy transfer properties of thesample fluid, wherein the region is sized to be substantially covered byan aliquot volume of the sample fluid; and a processing device coupledto the measurement device, the processing device configured to receivethe sample fluid reading and to process and directly determine theosmolarity of the sample fluid from the sample fluid reading.
 10. Anosmolarity measuring system as defined in claim 9, further includingnano-scale spheres of the sample region, wherein the nano-scale sphereshave a luminescence that is correlated with osmolarity of the samplefluid.
 11. An optical measuring system for measuring osmolarity of asample fluid, the system comprising: a sample-receiving chip comprisinga substrate adapted to receive the sample fluid, wherein the substrateincludes a sample region that is sized to be operatively covered by analiquot volume of the sample fluid; an optical energy source thatilluminates the sample region containing the sample fluid; and anoptical detector that receives optical energy from the illuminatedsample region in response to imparting energy into the sample fluid andprocesses the received optical energy to produce an electrical signalcomprising a sample fluid reading that is directly related to opticalproperties of the sample fluid and estimate the osmolarity of the samplefluid directly from the sample fluid reading.
 12. A system as defined inclaim 11, further comprising: volume-measuring elements to determine thealiquot volume of the sample fluid.
 13. A system as defined in claim 12,wherein the volume-measuring elements include a plurality of electrodes.14. A system as defined in claim 11, wherein the optical detectorincludes a photodiode.
 15. An optical measuring system as defined inclaim 11, wherein the sample region includes a plurality of nano-scalespheres having a luminescence correlated to osmolarity of the samplefluid.
 16. A method for determining osmolarity value of a sample fluidcomprising: depositing an aliquot volume of the sample fluid tooperatively cover a sample region of a substrate; imparting energy intothe sample fluid; producing an electrical signal comprising a samplefluid reading that is directly related to energy transfer properties ofthe sample fluid; and processing the sample fluid reading to directlydetermine the osmolarity value of the sample fluid.
 17. A method asdefined in claim 16, wherein measuring energy properties comprisesdetecting luminescence of a plurality of nano-scale spheres of thesample region, such that the luminescence is correlated to osmolarity ofthe sample fluid.
 18. A sample receiving chip comprising: a substratethat receives an aliquot volume of a sample fluid; a sample region ofthe substrate, sized such that the volume of the sample fluid issufficient to operatively cover a portion of the sample region,whereupon energy properties of the sample fluid can be detected from thesample region to produce a sample fluid reading, wherein the samplefluid reading indicates osmolarity of the sample fluid; wherein theregion includes a plurality of optical indicators disposed to contactthe sample, wherein the optical indicators comprise a plurality ofnano-scale spheres whose luminescence is correlated to osmolarity of thesample.
 19. An osmolarity measuring system for measuring osmolarity of asample fluid, the system comprising: a measurement device comprising asample receiving chip that includes a substrate having a sample regionconfigured to contact the sample fluid to measure energy properties ofthe sample fluid, wherein the region is sized to be substantiallycovered by an aliquot volume of the sample fluid; and a processingdevice coupled to the measurement device, the processing deviceconfigured to receive the measured energy properties and to process andestimate the osmolarity of the sample fluid from the processed energyproperties; wherein the measurement device includes a plurality ofnano-scale spheres that fluoresce with exposure to sample fluid ofvarying osmolarity; wherein the nano-scale spheres of the measurementdevice have a luminescence that is correlated to osmolarity of thesample.
 20. A method for determining osmolarity value of sample fluidcomprising: depositing an aliquot volume of the sample fluid on a sampleregion of a substrate; measuring energy properties of the sample fluid;and processing the measured energy properties to provide the osmolarityvalue of the sample fluid; wherein measuring the energy propertiesincludes: providing the substrate and sample region with appropriatethermal energy to solidify the sample fluid; detecting the temperatureat which the conductivity of the sample fluid changes in correlation tothe aforementioned solidifying as a result of the applied energy;wherein optical properties of the sample fluid change in accordance withchanges in osmolarity.
 21. A sample receiving chip comprising: asubstrate that receives an aliquot volume of a sample fluid; a sampleregion of the substrate, sized such that the volume of the sample fluidis sufficient to operatively cover a portion of the sample region,wherein energy imparted into the sample fluid is transduced by thesample region to produce an output signal that indicates energyproperties of the sample fluid that are correlated with osmolarity ofthe sample fluid.
 22. A sample receiving chip as defined in claim 21,wherein the imparted energy comprises electrical energy.
 23. A samplereceiving chip as defined in claim 22, wherein the sample regioncomprises a plurality of electrodes such that the received sample fluidcovers one or more of the electrodes and renders the electrodesconductive, and wherein the sample region further transduces theimparted electrical energy such that the output signal indicateselectrical energy properties of the sample fluid that are correlatedwith the osmolarity of the sample fluid; and wherein the sample regionfurther comprises nano-scale spheres that luminesce when exposed to thesample fluid and excited by the optical energy such that luminescence ofthe spheres is correlated to osmolarity of the sample fluid.
 24. Asample receiving chip as defined in claim 21, wherein the impartedenergy comprises optical energy.
 25. A sample receiving chip as definedin claim 24, wherein the sample region comprises optical indicators suchthat the received sample fluid covers a portion of the opticalindicators, wherein optical energy imparted into the sample fluid istransduced by the sample region to produce a luminescent signal thatindicates optical energy properties of the sample fluid that arecorrelated with osmolarity of the sample fluid.
 26. A sample receivingchip as defined in claim 25, wherein the optical indicators comprisenano-scale spheres.
 27. A sample receiving chip as defined in claim 26,wherein the nano-scale spheres comprise fluorescent nanobeads.
 28. Asample receiving chip as defined in claim 25, wherein the sample regionincludes a plurality of electrodes such that the received sample fluidcovers one or more of the electrodes, rendering the covered electrodesconductive, and wherein the electrodes are distributed across the sampleregion in an area of predetermined size, such that the number ofconducting electrodes determines the approximate volume of the samplefluid deposited on the substrate.
 29. A sample receiving chip as definedin claim 21, wherein the imparted energy comprises thermal energy.
 30. Asample receiving chip as defined in claim 29, wherein the thermal energycools the sample fluid to its freezing point such that the sample fluidfreezing point is observed and is correlated with osmolarity of thesample fluid.
 31. An osmolarity measuring system for measuringosmolarity of a sample fluid, the system comprising: a measurementdevice comprising a sample receiving chip that includes a substratehaving a sample region sized such that a received aliquot-sized volumeof the sample fluid is sufficient to operatively cover a portion of thesample region, wherein energy imparted into the sample fluid istransduced by the sample region to produce an output signal thatindicates energy properties of the sample fluid; a processing devicethat produces an osmolarity estimate of the sample fluid in accordancewith the output signal.
 32. An osmolarity measuring system as defined inclaim 31, wherein the imparted energy comprises electrical energy. 33.An osmolarity measuring system as defined in claim 32, wherein thesample region comprises a plurality of electrodes such that the receivedsample fluid covers one or more of the electrodes and renders theelectrodes conductive, and wherein the sample region further transducesthe imparted electrical energy such that the output signal indicateselectrical energy properties of the sample fluid that are correlatedwith the osmolarity of the sample fluid; and wherein the sample regionfurther comprises nano-scale spheres such that the received sample fluidcovers a portion of the nano-scale spheres, wherein optical energyimparted into the sample fluid is transduced by the sample region toproduce a luminescent signal that indicates optical energy properties ofthe sample fluid that are correlated with osmolarity of the samplefluid.
 34. An osmolarity measuring system as defined in claim 31,wherein the imparted energy comprises optical energy.
 35. An osmolaritymeasuring system as defined in claim 34, wherein the sample regioncomprises optical indicators that luminesce when exposed to the samplefluid and excited by the optical energy such that luminescence of theoptical indicators is correlated to osmolarity of the sample fluid. 36.An osmolarity measuring system as defined in claim 35, wherein theoptical indicators comprise nano-scale spheres.
 37. An osmolaritymeasuring system as defined in claim 36, wherein the nano-scale spherescomprise fluorescent nanobeads.
 38. A sample receiving chip as definedin claim 35, wherein the sample region includes a plurality ofelectrodes such that the received sample fluid covers one or more of theelectrodes, rendering the covered electrodes conductive, and wherein theelectrodes are distributed across the sample region in an area ofpredetermined size, such that the number of conducting electrodesdetermines the approximate volume of the sample fluid deposited on thesubstrate.
 39. An osmolarity measuring system as defined in claim 31,wherein the imparted energy comprises thermal energy.
 40. An osmolaritymeasuring system as defined in claim 39, wherein the thermal energycools the sample fluid to its freezing point such that the sample fluidfreezing point is observed and is correlated with osmolarity of thesample fluid.
 41. An optical measuring system for measuring osmolarityof a sample fluid, the system comprising: a sample-receiving chipcomprising a substrate adapted to receive the sample fluid, wherein thesubstrate includes a sample region that is sized to be operativelycovered by an aliquot volume of the sample fluid; an optical energysource that imparts optical energy into the sample fluid, wherein theoptical energy is transduced by the sample region and produces anoptical output signal that indicates energy properties of the samplefluid that are correlated with osmolarity of the sample fluid; and anoptical detector that receives the optical output signal from the sampleregion and processes the output signal to produce an estimate of thesample fluid osmolarity.
 42. An optical measuring system as defined inclaim 41, wherein the sample region comprises optical indicators thatluminesce in response to the optical energy such that luminescence ofthe optical indicators is correlated to osmolarity of the sample fluid.43. An optical measuring system as defined in claim 42, wherein theoptical indicators comprise nano-scale spheres.
 44. An optical measuringsystem as defined in claim 43, wherein the nano-scale spheres comprisefluorescent nanobeads.
 45. An optical measuring system as defined inclaim 42, wherein the sample region includes a plurality of electrodessuch that the received sample fluid covers one or more of theelectrodes, rendering the covered electrodes conductive, and wherein theelectrodes are distributed across the sample region in an area ofpredetermined size, such that the number of conducting electrodesdetermines the approximate volume of the sample fluid deposited on thesubstrate.
 46. An optical measuring system as defined in claim 41,wherein the imparted energy comprises thermal energy.
 47. An opticalmeasuring system as defined in claim 46, wherein the thermal energycools the sample fluid to its freezing point such that the sample fluidfreezing point is observed and is correlated with osmolarity of thesample fluid.